Purification and Characterization of Chitinase Produced by Endophytic Streptomyces hygroscopicus Against Some Phytopathogens

Microorganisms, which secret a complex o f mycolytic enzymes are considered to be possible bio logical control agents of plant d iseases. Since Chitinases are digestive enzymes that break down glycosidic bonds in chit in. In this present study, an industrial enzyme chitinase produced by endophyticStreptomyceswas purified and its antifungal act ivity was investigated against phytopathogens i.e. Rhizoctoniasolani,Fusariumoxysporum, Alternaria alternate, Aspergillusniger, Aspergillusflavus, Sclerotiniascleotiorum, Phytophthoraparasiticaand Botrytis cinerea.A chitinasewas produced byendophyticStreptomyceshygroscopicus in cultures containing chitin as the sole substrate that degrade chitin in 0.9 mm zone of clearance. The purification steps included ammonium sulfate precip itation, with columns of DEAE-Sepharose anion exchange chromatography and Sephacryl S-400 high resolution gel chromatography. The method gave a 5.4 fold increase of the specific activ ity and had a y ield of 18%. The molecular weight of the chit inase was found to be around 80.8, 78 and 76 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) technique. Chit inase was optimally active at pH of 6.0 to7.0 and at 30°C. The ch itanase were found to inhibit the growth of all phytopathogenic fungi.


Introduction
Fungal phytopathogens pose serious problems worldwide in the cultivation of economically important plants. Chemical fungicides are extensively used in current agriculture. However, excessive use of chemical fungicides in agriculture has led to deteriorating human health, environmental pollution, and development of pathogen resistance to fungicide. Because of the worsening problems in fungal disease control, a serious search is needed to identify alternative methods for plant protection, which are less dependent on chemicals and are mo re environ mentally friendly.
Microbial antagonists are widely used for the b iocontrol of fungal plant diseases [1]. Endophytic micro-organis ms have receiv ed cons iderab le atten tion fo r th eir poten tial as biocontrol agents of fungal p lant pathogens. Varied en zy me production may result in new b iochemical characteristics and in p art be respons ib le fo r th e inherent b iod ivers ity of endophytic micro -org an is ms. A mong the lyt ic en zy mes evaluated as a source of biocontrol agents, chitinases have been studied largely because these enzymes are produced by a variety of endophytic mic ro-organisms [2]. Endophyticactinomycetes have been cited as pro mising biocontrol agents, either acting directly on fungal cell walls or init iating increased plant responses against disease. In addition, research indicates that both mechanisms operate to control fungal pathogens [3]. Enzy me technology is an interdisciplinary field, and enzy mes are routinely used in many environ mental-friendly industrial sectors. With the advancement in biotechnology especially in the area of genetics, protein engineering, develop ments in bioinformat ics, and the availability of sequence data have opened a new era of enzy me applications in many industrial processes [4]. Ch itinase, a group of en zy mes capable of degrading chitin directly to low molecular weight product and found in a broad range of organis ms, including bacteria (Bacillus, Aeromonas, Pseudomonas, Serratia, Enterobacter, Actinomycete), fungi (Trichodermaand Aspergillus), and higher plants, insects, crustaceans, invertebrates and some vertebrates [5].
Chit inases have received increased attention due to their wide range of biotechnological applications, especially in the biocontrol of fungal phytopathogens [6]. Chit inasehave been implicated in plant resistance against fungal pathogens because of their inducible nature and antifungal activ ities in vitro. In viruses, chitinases are involved in pathogenesis [4].
Due to mult iple applications of chitinases in biocontrol, waste management, med icine and biotechnology they become interesting enzymes for study .

Micro-organism Strains and Growth Condi tions
Streptomyceshygroscopicuswas previously isolated fro m peanut plants and identified in Plant Pathology Depart ment, National Research Centre, Egypt. Cu lture was grown and maintained on solid starch mediu m [7]at 28℃.
The fungal pathogens Rhizoctoniasolani , Fusariumoxysp orum, Alternariaalternate, Aspergillusniger,Aspergillusflav us, , Sclerotiniascleotiorumand Botrytis cinereawere isolated originally fro m different naturally diseased plants collected fro m different agricultural fields. The isolated fungi were grown on potato dextrose agar (PDA) (Difco, USA) p lates and incubated at 28℃ for 4 to 6 days. Purification of the resulting isolates was done using the hyphal tip or single spore techniques to obtain them in pure cultures; the detected isolates were then transferred into slant of PDA and kept at 4℃ for fu rther studies. Pure cultures of the isolated fungi were identified according to the cultural properties, morphological and microscopical characteristics of each fungus [8].
Antag onistic acti vi ty assay: Dual culture assay was performed to assess the potential biocontrol activity of Streptomyceshygroscopicus against pathogenic fungi. In the dual culture assay, conidial suspension of each pathogen and bacterial suspension of Sterptomyceswere streaked on the surface of LB agar at a distance of 4 cm. The gro wth of both was examined daily for the format ion of inhibit ion zone and growth reduction.

Quantitati ve Determination of Extracellular Chitinase Acti vity
Sterptomyces was grown in ISP-2 broth [9]. with continuous shaking at 150 rp m at 27 ℃for 10 days. Cell-free supernatants were collected at 1-day intervals by centrifugation at 8,000 rp m for 20 min at 4 ℃. Cell pellets were d ried at 80 ℃ and weighed to determine the gro wth. Chit inase activity was quantitatively determined by measuring the reducing end group of N-acetylglucosamine (NA G)that degraded fro m colloidal ch itin as substrate [10]. The estimat ion of chit inase activity was carried out by the procedure described by Wang et al. [11].
Purificati on of chi tinase: The proteins fro m the culture were precip itated by ammon iu m sulphate (75%). The precipitate was collected by centrifuging at 8,000 × g fo r 20 min, and resuspended in an acetate buffer (50 mM, pH 5.0). It was dialyzed against the same buffer and freeze-dried. The sample was then loaded on a preequilibrated DEA E-cellu lose column (2 × 20 cm), and washed with the acetate buffer. The proteins were eluted in a stepwise gradient on NaCl (0-1.0 M ) at a flow rate of 24 ml/h. Fractions of 3 ml were collected, and the absorbance was read at 280 n m in a spectrophotometer. The fractions with chitinase activity were comb ined, dialy zed against the acetate buffer, and concentration by lyophilization. The concentrated sample was passed through a Sephadex G-100 colu mn (2 × 40 cm) and eluted with the acetate buffer at the rate of 15 ml/h. The fractions of 3 ml were collected, and the absorbance and chitinase activity were measured.
Chitinase acti vi ty measurements A fluoro metric assay was used to determine chitinase activity using 4-methylu mbelliferyl-N,N',N''-chitotriose (Sig ma, St. Louis, USA) as a substrate. The amount of 4 methylu mbelliferone (4-MU) released was measured spectrofluorometrically by using a fluorescence spectrophotometer (F-4500, Hitachi) (excitation 390 n m and emission 450 n m). One unit (U) of chitinase activity was defined as the amount of en zy me required to release one μmol of 4-M U per min at 37℃.

SDS-PAGE analysis
:Sodium dodecyl sulphatepolyacrylamid gel electrophoresis (SDS-PA GE) was performed according to Laemmli procedure [12]. using the Mini-Protean II apparatus (Bio-Rad, Hercu lus, USA). A 10% separating gel containing 0.01% g lycol ch itin as the substrate of chit inase was used to detect chitinase activity. After electrophoresis, proteins in the gel were renatured in 0.1 M sodium acetate buffer (pH 5.0) containing 1% Triton X-100 at 37℃. Subsequently, the gel was stained with 0.01% Calcofluor White M2R (Sig ma, St. Louis, USA) in 0.5 M Tris-HCl (p H 8.9) and examined for the chit inolytic bands under UV transillu minator [13]. In another gel, p roteins were stained with Coo massie Brilliant Blue G-250.
Protein concentration was determined by Bradford's method [14]using bovine serum albumin as standard.

The Studies of Chitinases Characterization Determinati on of Opti mum Te mperature and pH
The reaction was carried out at temperatures ranging fro m 20 to 80℃ and the ch itinaseactivity at different temperatures were measured to find the optimu m temperature. A similar method was applied at varying pH levels fro m 3.0 to 8.0 to determine the optimu m pH.

Inhi biti on of Fung al Growth by the Purified-Enzyme Extract
A spore suspension of pathogenic fungiwas uniformly spread on plates of PDA (potato dextrose agar). Discs that were soaked with the purified en zy me extract (5 U, 10 U) were laid on the seeded plates; the control was disc-soaked in boiled-enzy me ext ract. Fungal growth was observed over 2 d of incubation at 30℃.

Statistical Analysis
All data (ch itinase production and antagonistic isolates) were analysed for significance (P < 0.01) using the SAS software package.

The production of extracellular chitinase enzyme by a potent antagonist
The production of ext racellular chit inolytic en zy me in the strain was determined at different growth phases. The level of chit inase was sharply increased during the exponential phase and dramatically declined when the cells entered the stationary phase (Fig.2). The strain produced relatively high levels of chit inase (5.8 U/ mg p rotein) at day1 of the incubation period.

Purificati on of the chi tinase
The purificat ion procedures of the chitinase secreted by the S.hygroscopicus are summarized in Table 1. The results showed that, the enzyme was purified 1.9-fo ld with a specific activity of 38.7U/ mgprotein after ammoniu m sulfate fractionation. The enzy me was then purified with DEA E-sepharose fast flow and showed 4.7-fold en zy me purification with a specific act ivity of 7.5 U/ mg p rotein. The final purification step presented 5.4-fold enzy me purification with a specific activ ity of3.1 U/ mg protein. These results indicated that the effectiveness of the purificat ion method applied in this research and the chitinase was purified to 5.4-fold with a 18% yields ( Table 1).
The extract was purified through fractional precipitation with ammoniu m sulphate, column chro matography, and non-denaturing PAGE, and was then checked using SDS-PA GE. The result indicates high chitinase activity, which showed as a three band, at a mo lecular weight of about 80.8, 78 and 76 kDa (Fig.3).

Effect of temperature
The chitinase activity was most active at 30℃. Above 40℃, the activ ity decreased and was lost completely at 60℃. When the enzyme was kept at various temperatures for 30 min in an acetate buffer (pH 5.0), it was significantly inactivated above 50℃ and co mpletely at 65℃ (Fig. 4).

Effect of pH
The optimal pHs for ch itinase activity and stability of the chitinase were examined. The enzy me was most active between pH 5.0 and 6.0. It was relatively stable at pHs between 4.0 and 8.0, when kept at 4℃. However, beyond these pH ranges, it rapid ly lost its activity (Fig. 5). The activity assay was carried out at different temperatures using 1% (w/v) colloidal chitin (dissolved in 50 mM pH 7.2 Tris-HCl buffer) as substrate The activity assay was carried out at 60°C using 1% (w/v) colloidal chitin (dissolved in 50 mMdifferent pH buffer) as substrate   R.solani,F.oxysporum, A. alternate, A.niger, A.flavus, S. scleotiorumand B.cinerea growth was observed on agar plates with paper discs that were coated with the purifiedchitinaseenzyme (Fig. 6). Antifungal act ivity of the purified chitinase of Streptomyces was confirmed. The inhibit ion diameter of the 10 U enzy me was greater than that of the 5 U enzy me, and the boiled en zy me had no inhibitory activity against all pathogens.

Discussion
Microbial production of chitinase had great attention of both industrial and scientific environ ments, not only because of its wide spectrum of applications but also for the lacuna of an effective production method .Microorganis ms, which secret a complex o f mycolytic enzy mes are considered to be possible biological control agents of plant diseases.
Endophytes are micro-organis ms that inhabit p lant interior tissues. These microorganis ms cause no harm to the host and do not develop external structures, which exclude nodulating bacteria and mycorrh izal fungi as endophytic micro -organisms [15].
Chit inolyticactivity has been implicated in the biocontrol activity of several bacteria, including Streptomyces spp. Owing to the innocuous nature of these organisms, deliverysystems to introduce endophytic bacteria, such asStreptomyces spp., into plants have been developed, andthe potential for endophytes as biocontrol agents has beenexplo red [16]. In a recent study,  [1]. Shekharet al. [1]purified a bioactive compound from endophytic Streptomyces violaceusnigr that showed a strong antagonism towards various wood-rotting fungi, and chitinase enzymes wereassociated with this inhibition. In general, the higherchitinase activity was correlated with h igher fungalinhibit ion.
In the present study, the chitinolyticstrain, S.hygroscopicus showed high inhibition levels against fungi, and the fungal hyphae exhibited a degraded appearance after chitinolytic strain culture treat ment. In this study on chitinase production, extra cellular ch itinase activity was determined.The purificat ion steps included ammoniu m sulfate precipitation, with colu mns of DEA E-Sepharose anion exchange chromatography and Sephacryl S-400 high resolution gel chromatography on AKTA purifier 100 protein liquid chro matography. The method gave a 5.4 fo ld increase of the specific activ ity and had a yield of 18%.
En zy me production was increased inpotential phase and more amounts weredetected in the stationary growth phase.Chitinases arefairly stable over broad pH range.Chit inase was optimally active at p H of 6.0 to 7.0 and at 30 o C. The mo lecular weight of the chit inase was found to be around 80. 8 [17], it was postulated that chitinase produced by the antagonists could beinvolved in disease control. The production of theseenzymes was therefore used as the criteria forselection of potential biocontrol agents against pathogens.
Streptomycesstrains are a likely choice as potential biological controlagents. Antagonistic activity of several Streptomyces sp.against a number of fungal pathogenic species has beenknown for a long time [18 , 19 & 20]. Also, ingreenhouse experiments, Streptomyces sp. has conferredvarious degrees of protection on different plant speciesagainst soil-borne pathogenic fungi [2].Very high protein content of clamydospores ofFusariumsp. (7 -28%) may be responsible for their ability to resist lysis in soil.
Studies on chitinolytic microorganisms have yielded a large increase in knowledge regarding their role in inhib ition of growth of fungal plant pathogens (20 & 21). Still this knowledge is not sufficient enough to formu late a preparation based on these agents that can work efficiently in all different environmental conditions. Thebiocontrol agents are also affected due to the geological and environ mental conditions. Studying the environmental condit ions of the area where bio logical control agent has been employed and statistical based formulation approach techniques will increase life span of microorganisms in different ecological conditions.The results of this study led us to conclude that chitinase produced by endophytic Streptomyces has the potential for control of p lant pathogenic fungi. This study provides a quantitative assessment of Streptomyces chitinolytic and antifungal activ ities.